KR20230070306A - Whey protein compositions and methods for their preparation and uses thereof - Google Patents

Abstract

The present invention relates to the field of protein processing technology and discloses whey protein compositions, methods for their preparation and uses thereof. The whey protein composition of the present invention comprises whey protein and β-casein, wherein the mass ratio of β-casein to whey protein is at least 4.5:95.5. The whey protein compositions provided by the present invention consist primarily of novel whey protein ingredients that have slower digestibility than other currently marketed whey proteins. The whey protein composition is a whey protein concentrate rich in β-casein, to which micronized and complex natural polysaccharides can be added, and can achieve the function of delaying digestion to promote muscle synthesis and provide sufficient nutrition.

Description

Whey protein compositions and methods for their preparation and uses thereof

CROSS REFERENCES TO RELATED APPLICATIONS

[0001] This application is filed with the Chinese Intellectual Property Office on September 29, 2020, titled "WHEY PROTEIN COMPOSITION, AND PREPARATION METHOD THEREFOR AND USE THEREOF," Chinese Patent Application No. 202011068591.8, the entire contents of which are incorporated herein by reference.

technology field

[0002] The present disclosure relates to the field of protein processing technology, and more particularly to whey protein compositions and methods for their preparation and uses thereof.

[0003] Whey protein, called the king of proteins, is a protein extracted from milk. It is characterized by high nutritional value, easy digestion and absorption, and containing various active ingredients. It is recognized as one of the high quality protein supplements for the human body.

[0004] Among various proteins, whey protein has the highest nutritional value. Whey protein is a high-quality, complete protein and is also an animal protein. It contains 8 types of amino acids necessary for the human body in a reasonable ratio close to the ratio required by the human body. It is an indispensable substance for human life activities such as growth, development, and anti-aging. In addition, whey proteins are easily digested and absorbed, and many experimental studies have shown that ingestion of whey protein concentrates promotes humoral and cellular immunity, stimulates the human immune system, and prevents the development of chemically induced cancers. Proven. Whey protein is also low in fat and lactose, but contains β-lactoglobulin, α-lactalbumin, immunoglobulins and many other active ingredients. It is these active ingredients that give whey protein many health benefits that are beneficial to the human body. Therefore, it is considered as one of the high-quality protein sources required by the human body.

[0005] From a nutritional point of view, animal protein-derived foods contain components harmful to the human body, such as excessive saturated fatty acids and cholesterol, and when consumed excessively, body fat and cholesterol are likely to increase, thereby causing cardiovascular disease. can This problem can be avoided by consuming protein powder to supplement protein. In addition, protein powder is easy to ingest, has a high absorption rate, and can reduce the burden on the stomach. Therefore, taking protein powder is the best option for protein supplementation. Whey protein is the first thing people consider when choosing a protein powder.

[0006] Whey protein has a short time from oral intake to digestion and absorption and contains a large amount of branched chain amino acids such as valine, leucine, and isoleucine, which can generally prevent muscle fatigue and can be very effective in strengthening muscles. It is considered to be However, if this release property can be slowed down, it can better promote positive muscle synthesis, so it is widely used in many fields, such as prevention of muscle atrophy in the elderly, muscle recovery and weight loss after exercise in humans, and growth and development of children. .

[0007] The current state of sustained release whey is dependent on the development of "micelle whey" and an undisclosed polymerization process. The raw material WheyXR (WXR) sold by Glanbia is claimed to be a polymerized whey with sustained-release properties. It is believed that the large whey aggregates that form limit the ability of pepsin to reach the protein and have the ability to slow digestion in the stomach. Whey protein tends to move quickly through the stomach and into the small intestine, so more technical means are required for slow digestion.

[0008] It is therefore an object of the present invention to provide whey protein compositions that have slower digestion properties when passing through the gastrointestinal tract.

[0009] Another object of the present invention is to provide a whey protein composition capable of slowly releasing leucine.

[0010] Another object of the present invention is to provide a whey protein composition capable of delaying entry into the intestinal tract by significantly increasing the viscosity in the stomach.

[0011] Another object of the present invention is to provide a whey protein composition capable of reducing the digestibility of β-lactoglobulin.

[0012] Another object of the present invention is to provide a method for preparing the whey protein composition and the use of the whey protein composition in the manufacture of nutritional supplements, muscle synthesis promoters and dairy products.

[0013] In order to achieve the above object, the present invention provides the following technical solutions.

[0014] The whey protein composition comprises whey protein and β-casein, wherein the mass ratio of β-casein to whey protein is at least 4.5:95.5. The whey protein and β-casein used in specific embodiments of the present invention are primarily derived from cow's milk, but raw milk from other sources is not excluded. The whey protein composition of the present invention may be a qualified mixture prepared by direct processing of raw milk, or a mixture obtained by adding β-casein and whey protein in proportion.

[0015] Preferably, the mass ratio of β-casein to whey protein is (4.5:95.5)-(50:50); Further preferably the mass ratio of β-casein to whey protein is (9:91)-(20:80), more preferably the mass ratio of β-casein to whey protein is (9:90)-(17:83) )am. In a specific embodiment of the present invention, the mass ratio of β-casein to whey protein may be 4.6:95.4, 5:95, 9.4:90.6, 10:90, 15:85, 16.6:83.4 or 17:83.

[0016] In addition to directly mixing β-casein and whey protein, the present invention adds a specific treatment (including holding the cold for a period of time) to elute β-casein from the casein micelles and introduce it into the whey. can be used as Due to the unique molecular chaperone effect of β-casein, it forms a unique combination with α-lactalbumin and β-lactoglobulin in whey proteins, thereby protecting whey proteins from rapid digestion. The composition of the present invention, whether prepared by processing crude oil or blending and adding raw materials according to the ratio, is characterized by reducing the release rate of leucine compared to commercially available products.

[0017] In addition, as a result of analyzing β-lactoglobulin residues in compositions with different ratios of β-casein and whey protein, only compositions with a ratio of 4:96 or higher showed higher levels of β-lactoglobulin compared to standard whey protein concentrates. Digestion was found to be slower.

[0018] Based on the whey protein described above, the present invention achieves that the micronization process further enhances the sufficient binding of β-casein and whey protein, thereby further achieving the effect of slowly releasing leucine, and in vitro ( In vitro ) Digestion in the digestion model was found to increase the viscosity of gastric juice, which may prevent protein from being released into the intestine and delay the digestion process. Compared to the micronization process at pH 6.5, the micronization process at pH 7.5 has a more pronounced effect in this respect. In addition, the composition of the present invention, whether prepared by processing crude oil or added by mixing raw materials in a proportion, can obtain the above effects by pulverization. In addition, the micronization process can improve other physical properties of the mixture including particle size distribution, solubility, stability, viscosity, and the like. Surprisingly, the present invention found that if the standard whey protein concentrate only undergoes micronization without being combined with β-casein, the rate of digestion of the whey protein will be accelerated, suggesting that the addition of β-casein overcomes this excessively rapid digestion rate. It shows that you can change the phenomenon and slow down the digestion process further. Based on the above experimental results, the whey protein composition of the present invention is preferably a micronized whey protein composition.

[0019] Preferably, micronization is performed under high temperature and high shear at a pH value of 6.5-9.0; Here, the high temperature is 80-100 ° C, high-speed shearing is performed at 6000-20000 rpm, and the micronization time is 3-10 minutes. More preferably, the high temperature is 91-99 ° C, the high-speed shearing is performed at 8000-15000 rpm, and the micronization time is 4-8 minutes. Among them, the preferred parameters for the micronization process are pH 7.5, temperature 95°C, duration 5 minutes, and shear rate 9600 rpm. After micronization is complete, the materials are rapidly cooled to 15°C.

[0020] As a further solution optimization, the present invention adds a polysaccharide based on the above. Polysaccharides have more binding sites due to their inherent viscosity advantage and the presence of polyhydroxyl groups, thereby giving β-casein the ability to further bind to rich whey proteins, which further increases the viscosity of the mixture. may enhance or delay the rate of leucine release. Alginate and κ-carrageenan in the gastric juice of the in vitro digestion model have higher viscosities than other polysaccharides tested, where the viscosity of κ-carrageenan is 30-50 times that of other polysaccharides. Also, adding polysaccharide to micronization at pH 7.5 has a better effect than adding polysaccharide to micronization at pH 6.5.

[0021] According to the leucine release assay, potassium alginate can reduce the rate of release of leucine from micronized whey proteins rich in β-casein during in vitro digestion. Although the addition of κ-carrageenan did not significantly reduce the rate of release of leucine from β-casein-rich micronized whey proteins in the intestinal tract, given that κ-carrageenan can significantly increase the viscosity of gastric juice, κ-carrageenan After supplementation, it is expected that whey protein will pass more slowly into the intestinal tract, which will help delay the release of free leucine in the body's small intestine.

[0022] In digestion experiments, compared to standard whey protein concentrates and commercially available products and their polysaccharide-added controls, the micronized whey protein compositions of the present invention with the same added polysaccharides have lower hydration values than their respective controls. dissolution rate, which indicates that the whey protein composition of the present invention has a better sustained release effect. Similarly, the undifferentiated experimental group at pH 7.5 always has a better effect than the undifferentiated experimental group at pH 6.5.

[0023] Preferably, the polysaccharide of the present invention is a group consisting of carrageenan, alginate, chitosan, carboxymethyl cellulose, tragacanth gum and mixtures thereof is selected from, and the added amount of the polysaccharide is 0.1 to 0.3%. According to the above experimental results, in certain embodiments, κ-carrageenan and/or potassium alginate may be added to achieve superior effects in different digestive tracts.

[0024] In addition, the present invention is to process skim milk at a low temperature of 0 to 10 ° C (10 to 120 hours, preferably 10 to 24 hours), and then filter using a membrane of 20 to 200 nm or more. and concentrating the permeate by ultrafiltration to remove lactose to obtain a whey protein composition containing whey protein and β-casein. provided by

[0025] β-casein is released from the casein micelles and is concentrated in the supernatant of skim milk at low temperature. As the temperature decreases, the strength of the hydrophobic interaction decreases, preventing β-casein from freely eluting from the micelles and entering the supernatant. because it allows β-casein can form its own micelles with a diameter of 20-30 nm or less. Thus, a 20-200 nm (preferably 50 nm) ceramic or polymeric membrane is sufficient to retain the casein micelles while allowing whey proteins and β-casein to pass through and enter the permeate. Then, the permeate is concentrated through ultrafiltration (10 KDa) to remove lactose, and the remaining supernatant protein is β-casein and whey protein in a ratio of (4.5:95.5)-(20:80). include

[0026] SDS-PAGE gel electrophoresis results show that β-casein and whey protein obtained after processing milk at 10 ° C, 4 ° C and 2 ° C have the following ratios, using chemiluminescence imaging for evaluation : 4.6:95.4 at 10°C; 9.4:90.6 at 4°C and 16.6:83.4 at 2°C. Therefore, the production method of the present invention can easily obtain whey protein containing β-casein in an amount of 5 to 20%. If it is desired to achieve a whey protein composition comprising a higher percentage of β-caseincan, it can be obtained by compounding.

[0027] According to the description of beneficial effects obtainable by the micronization process, the manufacturing method of the present invention may further include a micronization process, wherein the micronization is carried out under high temperature and high-speed shear at a pH value of 6.5 to 9.0. This is followed by rapid cooling to prevent protein denaturation at high temperatures for a long period of time. Preferably, the high temperature is 80 to 100° C., the high-speed shearing is performed at 6000 to 20000 rpm, and the micronization time is 3 to 10 minutes. More preferably, the high temperature is 91 to 99 ° C, the high-speed shear is performed at 8000 to 15000 rpm, and the micronization time is 4 to 8 minutes. Among them, the preferred parameters for the micronization process are pH 7.5, temperature 95° C., dwell time 5 minutes, and shear rate 9600 rpm. After micronization is complete, the materials are rapidly cooled to 15°C.

[0028] Preferably, in the micronization process, concentration (protein concentration is 4% or more) to obtain a concentrate, followed by micronization treatment, and then further concentrating the resulting mixture so that the total solid content is 20 % or more, and then spray-drying the obtained material into a powder, converting the obtained material into a powder without spray drying, or making the obtained material into a composition in another form as necessary.

[0029] In certain embodiments of the present invention, the micronization process comprises concentrating the retentate after ultrafiltration at 55° C. until the protein concentration reaches 5%-10%, HCl to adjust the retentate to pH 7.5. or adding NaOH, processing the concentrate at 95°C for 5 minutes while performing high-speed shear at 9600 rpm, followed by rapid cooling of the micronized protein system to 15°C, followed by a total solids content of 20 This is done by further concentrating the resulting mixture to reach ˜25%, and finally spray drying the mixture at an inlet temperature of 200° C. and an outlet temperature maintained between 90-100° C.

[0030] After micronization, polysaccharide may be further added and mixed, and the polysaccharide is selected from the group consisting of carrageenan, alginate, chitosan, carboxymethyl cellulose, tragacanth gum, and mixtures thereof, which is in an amount of 0.1-0.3% is added as

[0031] According to the method of preparation provided above, the present invention further provides a whey protein composition prepared by the method. Since the whey protein composition provided by the present invention has a variety of slow digestion technology effects and has a slow digestion effect that is better than similar products on the market, the present invention provides for the preparation of whey protein formulations with slow digestion and/or slow release of amino acids. , further provided is the use of the whey protein composition. The product may be a nutritional supplement, muscle synthesis stimulant or dairy product.

[0032] Accordingly, the present invention further provides for the use of whey protein compositions in the manufacture of nutritional supplements, muscle synthesis promoters or dairy products. The whey protein composition may be a qualified blend prepared by processing raw milk, such as in the manufacturing processes mentioned above; or a mixture of β-casein and whey protein mixed in proportion; or mixtures to which additional micronized and/or polysaccharides have been added.

[0033] According to the above application, the present invention provides nutritional supplements, muscle synthesis promoters or dairy products. The names of the products are specifically determined according to the field of application, but as a common feature, they all contain the whey protein composition described in the present invention, and other ingredients, excipients and/or nutrients that can be added to foods according to the needs of the product. can be added with Nutrients include, but are not limited to, vitamins and minerals.

[0034] In a specific embodiment of the present invention, the present invention provides a nutritional supplement provided in the form of a nutritional bar, and in addition to the whey protein composition of the present invention, glucose syrup, glycerin, maltodextrin, coconut oil and lecithin.

[0035] The present invention more specifically also provides a dairy product, provided in the form of a powder, comprising the whey protein composition of the present invention and lactose, fructose, glucose, cream, vitamins, minerals and lecithin. Vitamins include, but are not limited to, vitamins A, C, D and E, and minerals include, but are not limited to, calcium, iron, zinc and magnesium salts, such as their phosphates or sulfates.

[0036] In digestion experiments of certain formula products, the present invention found that different formulas had a strong effect on the release rate of amino acids, wherein the digestion rate of leucine in the powder formulation was slower than that in the solid bar formulation. Under the same food formulation, the whey protein composition of the present invention has a leucine releasing effect close to that of the commercially available control sample.

[0037] It can be seen from the above technical solutions that the whey protein composition provided by the present invention is mainly composed of novel milk protein ingredients that have a slower digestibility than other whey proteins on the market. Micronized and blendable with natural polysaccharides is whey protein concentrate rich in β-casein, which can delay digestion, stimulate muscle synthesis and provide adequate nutrition.

[0038] Figure 1 shows an exemplary manufacturing flow diagram of the whey protein composition of the present invention;
[0039] Figure 2 shows protein electrophoretic graphs of whey protein compositions of the present invention obtained by treatment at different temperatures;
[0040] Figure 3 shows the leucine release curves of a whey protein composition rich in β-casein and a control after artificially digested with simulated digestive juices;
[0041] Figure 4 shows leucine release curves of a whey protein composition of the present invention and a control after artificially digested with simulated digestive juices;
[0042] Figure 5 shows viscosity curves when a whey protein composition of the present invention is formulated as a 5% protein concentrated solution and subjected to a micronization process;
[0043] Figure 6 shows viscosity curves of unmicronized whey protein compositions and control samples after being artificially digested with simulated digestive juices;
[0044] Figure 7 shows the viscosity curves of a whey protein composition of the present invention and a control sample after being artificially digested with simulated digestive juices;
[0045] Figure 8 shows viscosity curves after blending a whey protein composition of the present invention with alginate and carrageenan, respectively, and artificially digested with simulated digestive fluids;
[0046] Figure 9 shows a leucine release curve after combining a whey protein composition of the present invention with potassium alginate and artificially digested with simulated digestive fluids;
[0047] Figure 10 shows the leucine release curve after combining the whey protein composition of the present invention with carrageenan and artificially digested with simulated digestive fluids;
[0048] Figure 11 shows the whey protein retention curves in whey protein compositions of the present invention obtained by processing at different temperatures and artificially digested with simulated digestive juices;
[0049] Figure 12 is a histogram showing the relative proteolytic degradation of whey protein compositions of the present invention after digestion by simulated gastric and intestinal fluids, respectively.
[0050] Figure 13 is a histogram showing the degree of proteolysis of whey protein compositions of the present invention before and after digestion with artificial digestive juices.
[0051] Figure 14 shows a leucine release curve after a whey protein composition of the present invention is applied to a formulation and digested artificially with simulated digestive fluids.
15 shows non-reduced (NR) and reduced (R) gel electrophoresis images of whey protein compositions of the present invention.
[0053] Figure 16 shows the viscosity curves of whey proteins affected by different complex polysaccharides in artificially simulated digestive fluids.
[0054] Figure 17 shows the viscosity curves of whey protein affected by combining different concentrations of carrageenan in artificially simulated digestive fluids.

[0055] The present invention discloses whey protein compositions, methods of making and uses thereof. A person skilled in the art can learn from the contents herein and appropriately improve the process parameters to realize the present invention. In particular, it should be noted that all similar substitutions and modifications are obvious to those skilled in the art, and are all considered to be included in the present invention. The whey protein composition of the present invention and its manufacturing method and use have been described through preferred embodiments, and it is clear to those skilled in the art to implement and apply the technology of the present invention without departing from the content, spirit and scope of the present invention described herein. Modifications or appropriate changes and combinations may be made to the whey protein compositions and methods of making and using them.

[0056] Compared to regular whey protein concentrates and commercially available products, the main ingredients of the whey protein composition of the present invention include three improvements or any combination thereof: 1) low temperature concentration of β-casein by membrane filtration process at (0-10° C.); 2) improving properties such as particle size distribution, solubility, stability, viscosity, and leucine release by micronization treatment; 3) certain polysaccharides (kappa-carrageenan and/or potassium alginate) that do not cause gelation but further increase the viscosity and retard the release of leucine.

[0057] In the comparative experiments performed in the present invention, unless otherwise specified, additional experimental conditions remain the same except for significant differences between each group.

[0058] The whey protein composition provided by the present invention, its preparation method and its use are described in detail below.

Example 1: Preparation of whey protein composition using skim milk

[0059] Preparation of Whey Protein Ingredients Enriched with 1.β-Casein

[0060] The raw milk was degreased and sterilized, and then left at a low temperature such as 4° C. for 24 hours to release β-casein from casein micelles and concentrate it in the supernatant of skim milk. A 50 nm ceramic or polymeric membrane was used to retain casein micelles while allowing whey proteins and β-casein to pass through and enter the permeate. The permeate was then concentrated by ultrafiltration at 10 KDa to remove lactose. The remaining supernatant protein was whey protein material rich in β-casein. See FIG. 1 for an exemplary flow diagram.

[0061] 2. Undifferentiated

[0062] The residue from step 1 was concentrated at 55 °C to achieve a protein concentration of 5%. The concentrate was adjusted to pH 7.5 with 1M HCl or 40% NaOH and processed at 95° C. for 5 minutes under high shear at 9600 rpm. After this step, the micronized protein system was rapidly cooled to 15°C. The mixture was then further concentrated to reach a total solids content of 20-25% and finally spray-dried at an inlet temperature of 200°C and an outlet temperature maintained between 90-100°C.

3. Addition of polysaccharides

[0064] The optimal polysaccharide type and addition ratio for increasing the viscosity of the raw material in the gastric digestion stage were determined through experiments. 0.3% κ-carrageenan showed the maximum viscosity in the stomach, and 0.3% alginate also increased the viscosity and maintained the viscosity throughout the digestion process in the digestive tract. Finally, it was confirmed that the whey protein micronized at pH 7.5 and blended with the polysaccharide showed a superior sustained release effect different from whey protein concentrate.

Example 2: Whey protein source rich in β-casein obtained at different temperatures.

[0065] According to the procedure in step 1 of Example 1, milk was processed at temperatures of 10°C, 4°C and 2°C to obtain a whey protein source rich in β-casein. The ratio of β-casein to whey protein was estimated using chemiluminescence imaging. The results are shown in FIG. 2 .

The ratio is 4.6:95.4 at 10°C; 9.4:91.6 at 4°C; And it can be clearly seen in FIG. 2 that it is 16.6:83.4 at 2°C. Therefore, the present invention can easily obtain a whey protein composition having a ratio ranging from 4.5:94.5 to 17:83 at a low temperature of 2-10 ° C, and a whey protein composition having a ratio ranging from 4.5:94.5 to 20:80 at 0-10 ° C. One can expect to obtain a protein composition.

Example 3: In Vitro Digestion of β-Casein-Rich Whey Protein

[0067] Experimental purpose: To study the changes of β-casein protein and whey protein before and after digestion and to select protein sources with low leucine release.

[0068] Experimental method: The target protein was enzymatically digested using a related digestive enzyme according to a standardized static in vitro digestion method, and the leucine content in the digested solution was detected using a Shimadzu LCMS 2010 EV system.

[0069] Experimental grouping:

Control: WPC refers to whey protein concentrate (WPC392, Fonterra); WXR stands for WheyXR, which is commercially available.

[0071] Experiment 1: BCNWP6.5 represents a whey sample produced by membrane filtration containing β-casein when the pH was adjusted to 6.5 (prepared according to step 1 of Example 1);

[0072] Experiment 2: BCNWP7.5 represents a whey sample produced by membrane filtration containing β-casein when the pH was adjusted to 7.5 (prepared according to step 1 of Example 1);

[0073] Experiment 3: ComBCNWP7.5 represents a whey sample produced by mixing β-casein and whey protein WPC392 (10:90) when the pH was adjusted to 7.5;

The results are shown in Figure 3. Leucine in the two control samples reached 1.4–1.6 mM after 120–240 min of intestinal digestion, whereas the leucine released in the three experimental groups was only 0.2–0.4 mM after enzymatic hydrolysis in the intestinal digestion step. , indicating that the digestibility was lower in the three experimental groups.

Example 4: In Vitro Digestion of β-Casein-Rich Whey Protein

[0075] Experimental Objective: To compare the effect of β-casein protein on the digestibility of micronized and non-micronized whey protein.

[0076] Experimental method: According to the standardized static in vitro digestion method, the target protein was enzymatically digested using the relevant digestion enzyme, and the leucine content in the digested solution was detected using a Shimadzu LCMS 2010 EV system.

[0077] Experimental grouping:

Controls: WPC: Whey Protein Concentrate (WPC392, Fonterra);

Experiment 1: MWP6.5 represents micronized whey protein concentrate at pH 6.5;

Experiment 2: MWP7.5 represents micronized whey protein concentrate at pH 7.5;

[0081] Experiment 3: MBCNWP6.5 represents micronized β-casein rich whey raw material at pH 6.5 (prepared according to the method of Example 1, steps 1-2);

[0082] Experiment 4: MBCNWP7.5 represents micronized β-casein rich whey raw material at pH 7.5 (prepared according to the method of Example 1, steps 1-2);

[0083] Experiment 5: ComBCNWP7.5 represents a micronized β-casein enriched whey sample produced by mixing β-casein and whey protein WPC392 (10:90) at pH 7.5;

The results are shown in FIG. 4. The rate of leucine release in experimental groups 1 and 2 was faster than that in the control group, indicating that micronization can promote digestion of whey protein; In groups 3 and 4, digestibility was slower than in groups 1 and 2, indicating that β-casein can reduce the digestibility of micronized whey protein; The rate of amino acid release in Experiment 5 was similar to that of Experiments 3 and 4, suggesting that the slow-release effect of leucine could also be obtained by mixing β-casein and whey protein in proportions in Experiments 3 and 4.

Example 5: Viscosity testing of whey protein samples with different casein content

[0085] Experimental Objectives: Foods that are "concentrated" in gastric juice tend to delay gastric emptying and possibly significantly delay digestion. The purpose of this experiment is to determine which type of sample delays digestion by comparing the viscosity of the sample to a control.

[0086] Experimental method: A solution with a total protein content of 5% was prepared. After measuring the viscosity at a total protein concentration of 5% under the same conditions within 180 seconds using an MCR301 rheometer and a cone plate, a 2-hour digestion experiment was performed on simulated artificial gastric juice.

[0087] Experimental grouping:

Control: WPI6.5/7.5 (Whey protein isolate, Fonterra WPI895, same WPI in this experiment) represents micronized whey protein isolate at pH 6.5/7.5.

[0089] Experiment 1: 15/6.5 and 15/7.5 represent whey protein samples prepared by micronizing the sample solution at pH 6.5/7.5, respectively, prepared by holding skim milk at 15° C. for 24 hours, where β -The ratio of casein and whey protein was approximately 4:96.

[0090] Experiment 2: 10/6.5 and 10/7.5 represent whey protein samples prepared by micronizing the sample solution at pH 6.5/7.5, respectively, prepared by holding skim milk at 10° C. for 24 hours, where β - The ratio of casein and whey protein was approximately 5:95.

[0091] Experiment 3: 4/6.5 and 4/7.5 represent whey protein samples prepared by micronizing the sample solution at pH 6.5/7.5, respectively, by holding skim milk at 4°C for 24 hours, where β - The ratio of casein and whey protein was approximately 10:90.

[0092] The experimental results are shown in FIG. 5. Compared to the control sample WPI, the samples of the experimental group all had higher viscosity. The lower the storage temperature of the skim milk, the higher the viscosity of the prepared 5% protein solution. Comparing the two pH levels, the viscosity of the protein solution was higher at pH 7.5.

Example 6: Viscosity Experiments of Whey Protein Samples Enriched with β-Casein

Experimental purpose: To determine the effect of micronization on viscosity.

[0094] Experimental method: The viscosities of artificially simulated gastric and intestinal fluids were measured using an MCR301 rheometer and a cone-plate.

[0095] Experimental grouping:

Control 1: WPC (Whey Protein Concentrate, Fonterra WPC392); WXR stands for competing product WheyRX.

Control 2: MW represents whey micelles;

Control 3: MWP6.5 and MWP7.5A represent micronized whey protein WPC392 at pH 6.5 or 7.5, respectively;

[0099] Experiment 1: BCNWP6.5 and BCNWP7.5 represent whey samples containing β-casein produced by membrane filtration when the pH was adjusted to 6.5 and 7.5 respectively (by the method of step 1 of Example 1). prepared according to);

[00100] Experiment 2: MBCNWP6.5 and MBCNWP7.5 represent undifferentiated β-casein-rich whey protein at pH 6.5 or 7.5, respectively (prepared according to the method of steps 1-2 of Example 1);

[00101] The experimental results are shown in FIGS. 6 and 7. The viscosity of the gastric juice of the micronized experimental group 2 was significantly higher than that of the undifferentiated experimental group 1. In experimental group 2, the viscosity of the micronized material at pH 7.5 in gastric juice was significantly higher than that of the micronized material at pH 6.5.

Example 7: In vitro digestion experiment with polysaccharide raw material added - Viscosity test

[00102] Experimental Objective: To determine the effect of polysaccharides on viscosity increase.

[00103] Experimental method: The polysaccharide was added at a rate of 0.3% w/w, and the resulting mixture was mixed uniformly. The viscosities of artificially simulated gastric and intestinal fluids were detected using an MCR301 rheometer and cone-plate.

[00104] Experimental grouping:

[00105] Control 1: WXR+ALG represents commercial product WXR+potassium alginate;

[00106] Control 2: WXR+KCG represents the commercial product WXR+κ-carrageenan;

[00107] Experiment 1: MBCNWP6.5+ALG represents micronized whey rich in β-casein + potassium alginate at pH 6.5 (prepared according to the method of steps 1-3 of Example 1);

[00108] Experiment 2: MBCNWP7.5+ALG represents micronized whey rich in β-casein + potassium alginate at pH 7.5 (prepared according to the method of steps 1-3 of Example 1);

[00109] Experiment 3: MBCNWP6.5+KCG represents micronized whey rich in β-casein + κ-carrageenan at pH 6.5 (prepared according to steps 1-3 of Example 1);

[00110] Experiment 4: MBCNWP7.5+KCG represents micronized whey rich in β-casein + κ-carrageenan at pH 7.5 (prepared according to steps 1-3 of Example 1);

[00111] The experimental results are shown in FIG. 8. Experimental group 2 and experimental group 4 formed a complex with the polysaccharide at pH 7.5, which had a higher viscosity than that of the polysaccharide and that of the control group at pH 6.5.

Example 8: In vitro digestion experiment with polysaccharide raw material added - amino acid release

[00112] Experimental purpose: To determine the effect of the two screened on the rate of release of leucine.

[00113] Experimental method: The polysaccharide was added in a proportion of 0.3% w/w, and the resulting mixture was mixed uniformly. The target protein was enzymatically digested using the relevant digestive enzymes according to a standardized static in vitro digestion method, and the leucine content in the digested solution was detected with a Shimadzu LCMS 2010 EV system.

[00114] Experimental grouping:

Control 1: WPC (Whey Protein Concentrate, Fonterra WPC392);

[00116] Experiment 1: MBCNWP6.5 represents micronized β-casein rich whey at pH 6.5 (prepared according to Example 1 steps 1-2);

[00117] Experiment 2: MBCNWP7.5 represents micronized whey rich in β-casein at pH 7.5 (prepared according to steps 1-2 of Example 1);

[00118] Experiment 3: MBCNWP6.5+ALG represents micronized whey rich in β-casein + potassium alginate at pH 6.5 (prepared according to the method of steps 1-3 of Example 1);

[00119] Experiment 4: MBCNWP7.5+ALG represents micronized whey rich in β-casein + potassium alginate at pH 7.5 (prepared according to the method of steps 1-3 of Example 1);

[00120] Experiment 5: MBCNWP6.5+KCG represents micronized whey rich in β-casein + κ-carrageenan at pH 6.5 (prepared according to steps 1-3 of Example 1);

[00121] Experiment 6: MBCNWP7.5+KCG represents micronized β-casein rich whey +κ-carrageenan at pH 7.5 (prepared according to steps 1-3 of Example 1);

[00122] Experimental results show that potassium alginate can reduce the rate of amino acid release from micronized whey proteins rich in β-casein during in vitro digestion (FIG. 9).

[00123] Although the addition of κ-carrageenan could not significantly reduce the rate of release of leucine from micronized whey protein rich in β-casein in the intestinal tract (FIG. 10), κ-carrageenan produced a significant increase in the viscosity of gastric juice. Given this potential, one would expect whey protein to enter the intestine more slowly after supplementation with κ-carrageenan, which may help delay the release of free leucine from the small intestine in vivo.

Example 9: Digestion Experiment - Analysis of Residual β-Lactoglobulin in Whey Protein

[00124] Experimental purpose: To compare the degree of digestion and determine the ability of slow digestion.

[00125] Experimental method: In vitro digestion was performed after preparing a solution having a total protein content of 10%. Residual amounts of β-lactoglobulin before digestion, after digestion in gastric juice, and after digestion in intestinal juice were measured using size exclusion high-performance liquid chromatography (SCE-HPLC) and reversed-phase high-performance liquid chromatography (RP-HPLC). did

[00126] Experimental grouping:

[00127] Control: WPI6.5/7.5 (Whey Protein Isolate, Fonterra WPI895, same WPI in this test) represents micronized whey protein isolate at pH 6.5/7.5.

[00128] Experiment 1: 15/6.5 and 15/7.5 represent whey protein samples prepared by micronizing the sample solution at pH 6.5/7.5, respectively, by holding skim milk at 15° C. for 10 hours, where β- The ratio of casein and whey protein was about 4:96 (prepared according to the method of steps 1-2 of Example 1, changing temperature, cold treatment time and pH parameters accordingly).

[00129] Experiment 2: 10/6.5 and 10/7.5 represent whey protein samples prepared by micronizing the sample solution at pH 6.5/7.5, respectively, by holding skim milk at 10° C. for 10 hours, where β- The ratio of casein and whey protein was about 5:95 (prepared according to the method of steps 1-2 of Example 1, changing the temperature, cold treatment time and pH parameters accordingly).

[00130] Experimental Group 3: 4/6.5 and 4/7.5 were whey protein samples prepared by micronizing the sample solution at pH 6.5/7.5, respectively, by maintaining skim milk at 4° C. for 10 hours, wherein β-casein and The ratio of whey protein was approximately 10:90 (prepared according to the method of steps 1-2 of Example 1, changing the temperature, cold treatment time and pH parameters accordingly).

[00131] The experimental results are shown in FIG. 11. As the temperature decreased, the ratio of β-casein: whey protein increased, and the ratio of undigested β-lactoglobulin in whey protein gradually increased after in vitro digestion. This means that the experimental group with a higher percentage of β-casein showed a slower digestibility than the standard whey protein control group.

[00132] A whey protein composition with a ratio of β-casein to whey protein of about 5:95 (strictly 4.6:95.4) or β-casein to whey protein, compared to the residual amount of the standard whey protein controls (WPI/6.5 and WPI/7.5). Compositions with a higher percentage of whey protein may have more residual β-lactoglobulin at the end of digestion than the control. Thus, a whey protein composition of the present invention having a mass ratio of β-casein to whey protein greater than 4.6:95.4 may exhibit a slower digestibility.

Example 10: Digestion Experiment - Analysis of Degree of Hydrolysis 1

[00133] Experimental purpose: to compare the degree of hydrolysis and to determine the slow digestion capacity.

[00134] Experimental method: Using the method of J. Adler-Nissen (1979), the degree of hydrolysis of proteins digested with gastric juice and intestinal juice was tested.

[00135] Experimental grouping:

[00136] Control 1: WXR + ALG-G/I represents commercial WXR + 0.3% potassium alginate, each digested with simulated gastric/intestinal fluid.

[00137] Control 2: WXR+KCG-G/I represents commercial WXR + 0.3% κ-carrageenan, each digested with simulated gastric/intestinal fluid.

[00138] Experiment 1: MBCNWP6.5+ALG-G/I represents micronized whey protein rich in β-casein + 0.3% potassium alginate at pH 6.5 (prepared according to the method of steps 1-3 of Example 1) ), which was digested with simulated gastric and intestinal juices.

[00139] Experiment 2: MBCNWP7.5+ALG-G/I represents micronized whey protein rich in β-casein + 0.3% potassium alginate at pH 7.5 (prepared according to the method of steps 1-3 of Example 1) ), which was digested with simulated gastric and intestinal juices.

[00140] Experiment 3: MBCNWP6.5+KCG-G/I represents micronized whey protein rich in β-casein + 0.3% κ-carrageenan at pH 6.5 (prepared according to the method of steps 1-3 of Example 1) was digested), which was digested with simulated gastric and intestinal juices.

[00141] Experiment 4: MBCNWP7.5+KCG-G/I represents micronized whey protein rich in β-casein + 0.3% κ-carrageenan at pH 7.5 (prepared according to steps 1-3 of Example 1) ), which was digested with simulated gastric and intestinal juices.

[00142] The experimental results are shown in FIG. 12. The degree of hydrolysis in the gastric juice of the experimental groups 1 and 2 was about 30%, and the degree of hydrolysis in the gastric juice of the control group to which ALG was added exceeded 35%. In addition, the degree of hydrolysis in the intestinal fluid of the experimental groups 1 and 2 was about 40%, and the degree of hydrolysis in the intestinal fluid of the control group to which ALG was added was close to 55%. The degree of hydrolysis in the gastric juice of the samples of the experimental groups 3 and 4 was about 30%, and the degree of hydrolysis in the gastric juice of the control group to which KCG was added exceeded 35%. In addition, the degree of hydrolysis in the intestinal fluid of experimental groups 3 and 4 was about 40%, and the degree of hydrolysis in the intestinal fluid of the control group to which KCG was added was close to 50%. This indicates that the sustained-release effect of experimental groups 1, 2, 3 and 4 was more pronounced. The degree of hydrolysis of the micronized samples at pH 7.5 in experimental groups 1, 2, 3, and 4 was lower than that at pH 6.5, which means that the sustained-release effect of the micronized samples at pH 7.5 was better.

Example 11: Digestion Experiment - Hydrolysis Degree 2 Analysis

[00143] Experimental purpose: to compare the degree of hydrolysis and to determine the slow digestion capacity.

[00144] Experimental method: Using the trinitrobenzenesulfonic acid (TNBS) method introduced by J. Adler-Nissen (1979), the degree of hydrolysis of proteins digested with gastric juice and intestinal juice was tested.

[00145] Experimental grouping:

[00146] Control 1: WPC (Whey Protein Concentrate, Fonterra WPC392);

[00147] Control 2: WXR (WheyRX, competitor);

[00148] Experiment 1: MBCNWP6.5+ALG represents micronized whey protein rich in β-casein + 0.3% potassium alginate at pH 6.5 (prepared according to the method of steps 1-3 of Example 1), which It is decomposed with artificial simulated digestive juices.

[00149] Experiment 2: MBCNWP7.5+ALG represents micronized whey protein rich in β-casein + 0.3% potassium alginate at pH 7.5 (prepared according to the method of steps 1-3 of Example 1), which It is decomposed with artificial simulated digestive juices.

[00150] Experiment 3: MBCNWP6.5+KCG represents micronized whey protein rich in β-casein + 0.3% κ-carrageenan at pH 6.5 (prepared according to the method of steps 1-3 of Example 1); It is decomposed into artificial simulated digestive juice.

[00151] Experiment 4: MBCNWP7.5+KCG represents micronized whey protein rich in β-casein + 0.3% κ-carrageenan at pH 7.5 (prepared according to the method of steps 1-3 of Example 1); It is decomposed into artificial simulated digestive juice.

[00152] The experimental results are shown in FIG. 13. The degree of hydrolysis of the samples of Experimental Groups 1, 2, 3 and 4 did not exceed 41%, the degree of hydrolysis of Control 1 exceeded 44%, and the degree of hydrolysis of the samples of Control 2 exceeded 42%. This indicates that 3 and 4 had a relatively prominent sustained-release effect. The degree of hydrolysis of the micronized samples at pH 7.5 in experimental groups 1, 2, 3, and 4 was lower than that at pH 6.5, which means that the sustained-release effect of the micronized samples at pH 7.5 was better.

Example 12: Digestion Experiment After Addition of Sample to Formulation - Amino Acid Release

[00153] Experimental purpose: To determine the effect of different formulations on digestion rate.

[00154] Experimental method: The final product was prepared according to the formula shown in Table 1 below, and the target protein was enzymatically digested using a related digestive enzyme according to the standardized static in vitro digestion method, and the content of leucine in the digested solution was Shimadzu ( Shimadzu) LCMS 2010 EV system.

Powder (per 100 g) P1 Solid bar (per 60g) P2 ingredient content (%) ingredient content (%) Protein (MBCNWP7.5 /WXR) 28.1 Protein (MBCNWP7.5/ WXR) 46 κ-carrageenan 0.3 glucose syrup 27 lactose 26.3 glycerin 17 Saccharides (42% fructose + glucose) 26.6 Maltodextrin, DE to 17 3.5 fat (cream) 10 coconut oil 5 vitamin
vitamin C
Vitamin E (as Tocopherol)
vitamin A
vitamin D One κ-carrageenan 0.3 mineral:
calcium phosphate
ferrous sulfate
zinc sulfate
magnesium sulfate 3 lecithin 0.5 lecithin 0.5 water 0.7 water (in powder) 4-5

[00155] Experimental grouping:

[00156] Control 1: P1+WXR refers to commercial product WXR slow release powder formulation P1 + whey ingredient;

[00157] Control 2: P2+WXR refers to solid bar formulation P2 + slow release commercial product WXR whey ingredient;

[00158] Experimental Group 1: P1+MBCNWP7.5 represents powder formulation P1+micronized whey source rich in β-casein+0.3% κ-carrageenan at pH 7.5 (prepared according to steps 1-3 of Example 1) );

[00159] Experimental group 2: P2+MBCNWP7.5 represents a solid bar formulation + micronized β-casein rich whey raw material at pH 7.5 + 0.3% κ-carrageenan (prepared according to steps 1-3 of Example 1) ).

[00160] The experimental results are shown in FIG. 14. Different food formulations have a great influence on the release rate of amino acids, and the digestion rate of powder formulations is slower than that of solid bar formulations; Under the same food formulation, the whey protein composition of the present invention exhibited a similar leucine release effect as the WXR control sample.

Example 13: Non-reduced (NR) and reduced (R) SDS-PAGE gel electrophoresis experiments of different protein samples

[00161] Experimental grouping:

[00162] 1. milk;

[00163] 2. Whey protein isolated from milk of group 1;

[00164] 3. Whey protein rich in β-casein prepared from whey proteins of group 2 (retentate after ultrafiltration/diafiltration, total solids concentration 0.2%);

[00165] 4. Micronized whey protein at pH 6.5 (same as whey protein of group 2);

[00166] 5. Micronized whey protein at pH 7.5 (same as whey protein of group 2);

[00167] 6. Micronized β-casein rich whey protein at pH 6.5 (same as group 3 whey protein, total solids concentration is 10%);

[00168] 7. Micronized β-casein rich whey protein at pH 7.5 (same as group 3 whey protein, total solids concentration is 10%);

[00169] 8. Whey protein rich in β-casein micronized at pH 6.5 (same as group 6, powder);

[00170] 9. Whey protein rich in β-casein micronized at pH 7.5 (same as group 7, powder).

[00171] The results are shown in FIG. 15. Non-reducing (NR) gel electrophoresis shows only proteins that are not aggregated with other proteins, while reducing (R) gel electrophoresis shows all proteins because the aggregation reaction between proteins is destroyed. Lanes 4, 6 and 8 represent micronized protein at pH 6.5, and the bottom band (representing α-lactalbumin) in the non-reducing gel was almost identical to lane 3 of the non-micronized control sample. Also, the second band from the bottom (representing β-lactoglobulin) almost disappeared, indicating that β-lactoglobulin was almost completely aggregated after undifferentiation.

[00172] Lanes 5, 7 and 9 show undifferentiated protein at pH 7.5 where the bottom band became obscured, and the second band was slightly more intense than the bands in lanes 4, 6 and 8, indicating more α-lactalbumin and indicating that less β-lactoglobulin was aggregated. Thus, different micronization mechanisms of whey proteins at the two pHs can be observed in this figure.

Example 14: Viscosity test of whey protein artificially added with different polysaccharides to simulated digestive juices

[00173] In simulated gastric fluid (without enzymes), the viscosity of WPC392(WP) protein solutions with a protein concentration of 8% and 0.2% or 0.3% by weight of the selected polysaccharide was added was detected. ALG stands for potassium alginate; CMC stands for carboxymethyl cellulose; TGH stands for tragacanth gum; XTH represents xanthan gum, Temp represents temperature, and GJ represents artificial gastric juice. The results are shown in FIG. 16 .

[00174] In addition, the viscosity of the WPC392 protein solution in which the protein concentration was 8% and carrageenan was added at 0.1-0.3% by weight in artificial gastric juice (no enzyme) was detected. Pure represents 0.1% by weight of carrageenan. The results are shown in FIG. 17 .

[00175] From Figures 16 and 17, it can be clearly seen that the viscosity of κ-carrageenan in the gastric juice of the in vitro digestion model was significantly higher compared to that of the other tested polysaccharides, which was about 30- It was 50 times. The viscosity of potassium alginate in the gastric juice of the in vitro digestion model was also significantly higher than that of the other polysaccharides tested.

[00176] It should be noted that the above is only a preferred embodiment of the present invention, and several improvements and modifications can be made by those skilled in the art without departing from the principles of the present invention, and these improvements and modifications fall within the protection scope of the present invention. should be regarded as

Claims (17)

A whey protein composition comprising whey protein and β-casein,
A whey protein composition wherein the mass ratio of β-casein to whey protein is at least 4.5:95.5. The whey protein composition of claim 1 , wherein the mass ratio of β-casein to whey protein is from 4.5:95.5 to 50:50. 3. The whey protein composition of claim 1 or 2, wherein the whey protein composition is a micronized whey protein composition. 4. The whey protein composition of claim 3, wherein the micronization is performed at a pH of 6.5-9.0 under high temperature and high shear. 5. The whey protein composition according to any one of claims 1 to 4, further comprising a polysaccharide. 6. The whey protein composition of claim 5, wherein the polysaccharide is selected from the group consisting of carrageenan, alginate, chitosan, carboxymethyl cellulose, gum tragacanth, and mixtures thereof. Processing skim milk at a low temperature of 0 to 10 ° C,
then performing filtration using a membrane of at least 20-200 nm; and
Concentrating the permeate by ultrafiltration to remove lactose to obtain a whey protein composition containing whey protein and β-casein.
Method for producing a whey protein composition comprising a. 8. The method of claim 7 further comprising micronizing the whey protein composition. The method of claim 8, wherein the micronization is performed at a pH of 6.5 to 9.0 under high temperature and high shear. 10. The method of any one of claims 7-9, further comprising adding a polysaccharide. 11. The method of claim 10, wherein the polysaccharide is selected from the group consisting of carrageenan, alginate, chitosan, carboxymethyl cellulose, gum tragacanth, and mixtures thereof. A whey protein composition prepared by the method according to any one of claims 7 to 11. Use of the whey protein composition according to any one of claims 1 to 6 or the whey protein composition according to claim 12 for use in the manufacture of nutritional supplements, muscle synthesis stimulators or dairy products. Use of the whey protein composition according to any one of claims 1 to 6 or the whey protein composition according to claim 12 for use in the manufacture of a whey protein product having slow digestion and/or slow release of amino acids. A nutritional supplement, muscle synthesis promoter or dairy product comprising the whey protein composition according to any one of claims 1 to 6 or the whey protein composition according to claim 12 and excipients and/or nutrients which can be added to food. 16. A nutritional bar according to claim 15 comprising the whey protein composition according to any one of claims 1 to 6 or the whey protein composition according to claim 12, and glucose syrup, glycerin, maltodextrin, coconut oil and lecithin. (bar), nutritional supplements, muscle synthesis promoters or dairy products. 16. A powder according to claim 15 comprising the whey protein composition according to any one of claims 1 to 6 or the whey protein composition according to claim 12 and lactose, fructose, glucose, cream, vitamins, minerals and lecithin. Nutritional supplements, muscle synthesis stimulants or dairy products, which are dairy products.

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